Wildtype barley seed contains approximately 50 to 60% of starch, contained in its endosperm, that has approximately 25% amylose and 75% amylopectin. Amylose is a mostly linear α-(1-4) linked glucosyl chain with a few α-(1-6) linked glucan chains and has a molecular weight of 104 to 105. Amylopectin is a highly branched glucan in which α-(1-4) linked glucosyl chains with mostly 3 to 60 glucosyl units are connected by α-(1,6)-linkages, so that approximately 5-6% of the glucosyl linkages are α-(1,6)-linkages, and has a molecular weight of 105 to 106.
A suite of enzymes are involved in cereal starch biosynthesis including ADP-glucose pyrophosphorylases (EC 2.7.7.27), starch synthases (EC 2.4.1.21), starch branching enzymes (EC 2.4.1.18) and starch debranching enzymes (EC 3.2.1.41 and 3.2.1.68). The first committed step of starch synthesis is synthesis of ADP-glucose from Glucose-1-P and ATP, catalysed by the enzyme ADP-glucose pyrophosphorylase. The ADP-glucose is then used as substrate for the synthesis of starch by starch synthases which transfer glucose to the non-reducing end of pre-existing α-(1-4) linked glucosyl chain of starch. The branched glucan chains of starch, linked with α-(1-6) linkages, are formed by starch branching enzymes through the cleavage of a region of the α-(1-4) linkage glucan and subsequent transfer of the short glucan to a position on the α-(1-4) linkage glucan of starch. Excess α-(1-6) linked glucan chains are removed by debranching enzymes to maintain starch in a defined structure (See reviews from Kossmann and Lloyd, Grit Rev Plant Sci, 19: 171-226, 2000; Rahman et al., J Cereal Sci, 31: 91-110, 2000; Smith, Biomacromolecules, 2: 335-341, 2001; Morell et al., Euphytica, 119: 55-58, 2001; Morell et al., J Appl Glycosci, 50: 217-224, 2003a; Morell et al., Control of starch biosynthesis in vascular plants and algae. In: Plaxton W C, McManus M T (eds) Control of primary metabolism in plants. Annual plant reviews, vol 22, Blackwell, Oxford, pp 258-289, 2006; Ball and Morell, Annu Rev Plant Biol, 54: 207-233, 2003; James et al., Curr Opin Plant Biol, 6: 215-222, 2003; Tetlow et al., J Exp Bot, 55: 2131-2145, 2004).
Ten starch synthase genes have been identified in the rice genome (Hirose and Terao, Planta, 220: 9-16, 2004) and are grouped into five distinct classes: granule-bound starch synthase (GBSS), starch synthase I (SSI), starch synthase II (SSII), starch synthase III (SSIII) and starch synthase IV (SSIV) (Li et al., Funct Integr Genomics, 3: 76-85, 2003). There are two GBSS isoforms (GBSSI and GBSSII), one SSI isoform, three SSII isoforms (SSIIa [SSII-3], SSIIb [SSII-2], and SSIIc [SSII-1]), two SSIII isoforms (SSIIIa [SSIII-2] and SSIIIb [SSIII-1]), and two SSIV isoforms (SSIVa [SSIV-1] and SSIVb [SSIV-2]) in rice (Hirose and Terao, 2004 (supra); Fujita et al., Plant Physiol, 144: 2009-2023, 2007). Proteins corresponding to SSI, SSIIa and GBSSI have been detected within starch granules, whereas SSIIIa protein has been only detected in the soluble phase of amyloplastids (Li et al., Plant Physiology, 123: 613-624, 2000). The precise role of these starch synthases individually and cooperatively in determining the final structure of the starch granule largely remains undefined although the potential roles of some starch synthases have been characterised in different organs and different species.
Mutants in starch synthases have been useful in determining the roles in some cereal species. GBSSI plays a crucial role in the biosynthesis of amylose (Ball et al., Cell 86(3): 349-52, 1996), but it may also contribute to the synthesis of the long chains of amylopectin (Maddelein et al., J Biol Chem. 269(40): 25150-7, 1994; Denyer et al., Plant Physiol. 112(2):779-85, 1996). The effect on starch properties has been examined for GBSSI null mutants in barley and wheat (Andersson et al., J Cereal Sci 30: 183-191, 1999; Yamamori and Quynh, Theor Appl Genet, 100: 32-38, 2000). The GBSSI null mutant barley had less than 5% of the amylose content compared to wild type (Andersson et al., 1999 (supra)). A GBSSI null mutant of wheat also had low amylose content (Kim et al., J Cereal Sci, 37: 195-204, 2003; Miura et al., Euphytica, 108: 91-95, 1999; Miura et al., Euphytica, 123: 353-359, 2002). The GBSSI null mutant wheat also had higher peak gelatinization temperature and enthalpy than that from wildtype as determined by Differential Scanning calorimetry (DSC) (Yasui et al., J Cereal Sci, 24: 131-137, 1996).
SSI, SSIIa and SSIII are thought to be primarily involved in amylopectin synthesis involved in the extension of specific subsets of available non-reducing ends within the starch molecule. Studies on Arabidopsis and rice SSI null mutants showed that SSI is involved in biosynthesis of the small outer chains of the amylopectin cluster (8-12 dp) in leaf starch of Arabidopsis (Delvalle et al., Plant J 43(3): 398-412, 2005) and in the endosperm starch of rice (Fujita et al., Plant Physiol. 140: 1070-1084, 2006). Starch from barley and wheat SSIIa mutants had an increase in chains of DP3-8, indicating that the SSIIa enzyme played a role in extending shorter glucan chains of DP3-8 to longer glucan chains of DP12-35 (Morell et al., Plant J. 34: 173-185, 2003b; Yamamori et al., Theor Appl Genet, 101: 21-29, 2000; Konik-Rose et al., Theor Appl Genet, 115: 1053-1065, 2007). Loss of SSIIIa in maize and rice conferred an increased amylose phenotype, with a reduction in the proportion of very long chains (DP>50 in maize or DP>30 in rice), and slightly reduced gelatinisation temperature (Jane et al., Cereal Chem. 76: 629-637, 1999; Fujita et al., 2007 (supra)). Arabidopsis mutants, defective for SSIV, appear to have fewer, larger starch granules within the plastid and a role in priming starch granule formation has been postulated for the SSIV protein (Roldan et al., Plant J 49: 492-504, 2007).
A barley SSIIa mutant has been shown to have a high amylose phenotype with reduced starch content and reduced seed weight due to the reduction of starch biosynthesis. The mutant barley lines M292 and M342 which were homozygous for a null mutation in the gene encoding SSIIa were obtained following mutagenesis of grains of the barley variety ‘Himalaya’ with sodium azide. Mutant seeds were initially selected from progeny grain of the mutagenised population on the basis of a shrunken grain phenotype. The mutant lines were further characterised by their altered starch properties, reduced SSIIa protein level and activity, and genetically by the presence of a premature stop codon in the protein coding region of the gene encoding SSIIa (Morell et al., 2003b (supra) incorporated herein in its entirety by reference). This caused loss of the SSIIa enzyme in the endosperm. However, the SSIIa mutant grain also had substantially reduced starch content and this was associated with a moderate reduction in yield when the barley plants were grown in the field. It was not known if the yield could be improved, or how, while still maintaining the high amylose phenotype.
There is therefore a need for high amylose barley with improved agronomic performance.